Dengue viruses (DENV), members of the Flavivirus family of viruses, cause periodic explosive epidemics in many tropical and sub-tropical countries leading to 50-100 million infections per year [World Health Organization (2012)]. Approximately 500,000 of these are severe cases requiring hospitalization with a 2.5% fatality rate, most of which are children [Randolph et al. (2010)]. About half the world's population remains at risk for DENV infection making this pathogen one of the most dangerous viruses in the world [Clyde et al. (2006)]. In 2010 there were 1.6 million cases of dengue in the Americas alone, of which 49,000 were severe cases. Recent domestic outbreaks have occurred in the Hawaiian Islands in 2001, Brownsville, Tex. in 2005 [Ramos et al. (2008)], the Florida Keys in 2010, and other parts of southern Florida including Miami-Dade in 2011 [Anez et al. (2012); Adalja et al. (2012); Effler et al. (2005); World health Organization (2012)]. Devastating outbreaks continue to occur in Puerto Rico, Brazil, and Pakistan [Anez et al. (2012); Figueiredo et al. (2012); Rai (2011)].
CHIKV remained largely unknown until a series of large scale epidemics occurred on several islands in the Indian Ocean in 2005 and 2006 culminating in a catastrophic outbreak on the island of la Reunion, resulting in 265,000 infections and 237 deaths in a population of 775,000 [Tsetsarkin et al. (2006)]. CHIKV has since been imported into Europe by infected travelers returning from endemic areas as evidenced by a CHIKV introduction in the French Riviera [Cordel et al. (2006)]. Most recently, CHIKV outbreaks have occurred and are currently ongoing on multiple Caribbean Islands including St. Maarten, British Virgin Islands, Guadeloupe, Martinique, Saint Barthelemy, and French Guiana [Van Bortel et al. (2014)]. These statistics, coupled with the worldwide distribution of Aedes aegypti and Aedes albopictus mosquitoes, demonstrate a risk of importing CHIKV into new areas, including the United States[Thiboutot et al. (2010)], through infected travelers.
The CHIKV outbreaks on La Reunion Island are believed to have been primarily facilitated by an Ala to Val (E1 A226V) amino acid substitution in the CHIKV glycoprotein E1 [Tsetsarkin et al. (2009)]. This mutation allowed the virus to traverse the A. albopictus gut membrane barrier more efficiently, resulting in a greater degree of dissemination through local swarms [Tsetsarkin et al. (2006)]. This likely provided a selective advantage for A. albopictus over Ae. aegypti as the insect vector, which accelerated the transmission of CHIKV to an immunological naïve population on la Reunion Island [Tsetsarkin et al. (2009)].
DENV are maintained in a cycle that involves humans and the globally disseminated Aedes aegypti mosquito [Roberts et al. (2002)]. Infection with one of four antigenically-distinct, but genetically-related DENV serotypes (designated DENV-1, -2, -3, and -4) can result in dengue fever (DF), dengue hemorrhagic fever (DHF), which can be fatal, or both DF and DHF [Qi et al. (2008)]. These disease states are characterized by high fever, often with enlargement of the liver, and in severe cases, circulatory and respiratory failure [Rigau-Perez et al. (1998)].
While DF and DHF are endemic to tropical and subtropical regions of the world, collapse of effective vector control programs, rapid dispersal of viruses due to ease of global travel, and migration of humans from tropical to non-tropical regions has resulted in DENV outbreaks in regions that were once non-endemic to these viral pathogens.
The ability to detect DENV in a timely manner is essential to rapid recovery from disease symptoms. Detection of mosquito-borne viruses in infected humans is currently limited to plaque assays, antigen detection assays (e.g. NS1 antigen detection), or quantitation of viral production through PCR-based methods [Lanciotti et al. (1992); Gubler (1998)]. These assays are currently referred to as the “gold standards” for DENV detection [de Oliveira et al. (2005)]. Current methods of testing mosquito populations for arboviruses, particularly dengue and West Nile viruses, has been limited to RT-PCR assays on pools of mosquitos (approximately 50 insects) [Shu et al. (2004); Chisenhall et al. (2008)].
The approaches mentioned above are limited by a number of pitfalls including low-throughput, labor-intensiveness, low stability of assay components at or above room temperature, and lack of portability. The requirement for specialized training and equipment and the time consuming nature of these assays limits their widespread utility for virus detection. These limitations compromise rapid diagnosis of viral infections. These methods are not easily adapted to field environments where reliable and effective detection methods are needed. Rapid, low-tech virus detection methods that require no specialized training or education are sorely needed to provide remote areas of the world the ability to detect highly pathogenic viruses for both clinical diagnosis and epidemiological surveillance.
In this report, we describe the development and initial validation of a colorimetric method for detecting DENV that couples the RNA targeting ability of a DENV-specific DNAzyme (DDZ) with the aggregation properties of oligonucleotide-tethered, non-crosslinking gold nanoparticles (AuNPs). Our new DENV detection system, called DDZ-AuNP (FIG. 1), should be an invaluable tool for the detection of DENV since it solves many of the limitations of current virus detection assays. This assay and subsequent analysis is cost-effective, simple to perform, and the assay components are highly stable at temperatures above 30° C., enabling easy storage at room temperature. The use of DNAzymes in the assay increases the specificity and versatility of detection permitting the design and incorporation of additional virus or strain-specific DNAzymes and probes.
Full development of this detection assay would greatly enhance virus diagnostics and epidemiology by providing an assay that is more rapid, easier to use, has greater portability, and is more cost effective than current DENV detection methods.